JP2016531131A - Production of probiotics by in vitro enrichment of beneficial microorganisms from human or animal microflora - Google Patents

Abstract

The present invention relates to a method of treating enterotoxiosis in a mammal, preferably a human, by taking a sample of the microorganism responsible for enterotoxiosis. It is done after the sample has been replaced and after the sample has been concentrated in vitro for healthy bacteria. [Selection figure] None

Description

  The present invention relates to the fields of food, medicine, bacteriology and health science. More specifically, it relates to the field of bacteria-based health foods and / or treatments by administration of bacterial cultures.

  Probiotics are defined by FAO and WHO as living microorganisms that, when administered in sufficient amounts, give a beneficial health effect to the host. In most cases it is administered orally, but the definition does not exclude other modes of administration. The addition of probiotic bacteria to food is an approach for improving the health of individual humans or animals by promoting the presence of beneficial bacteria in the intestine.

  The role that probiotics have in the microorganism-microbe interaction includes other competition for nutrients and colonization with harmful microorganisms, including pathogenic bacteria such as Clostridium difficile, Clostridium perfringens, E. coli, and the like. Lobiotics including acidification can function by the production of antibacterial substances such as bacteriocin and / or creation of restricted physiological states caused by, for example, lactic acid and other fermentations (1).

  The use of probiotics as a dietary intervention, in part, can cause certain strains selected from healthy intestinal flora to have strong anti-pathogenic and anti-inflammatory properties and therefore against intestinal diseases This is based on the concept that resistance can be provided (Non-Patent Document 2). In monogastric animals, including humans, the commensal microflora contributes to gut protection against pathogens by competing for nutrients and pathogen binding sites and / or modulating immune responses.

  Many probiotic compositions have been known since early in history. The earliest probiotics are components of products that we currently recognize as “normal” fermented foods such as yogurt, tempeh, juice and soy beverages. An overview of medically used probiotics has been provided by Non-Patent Document 3.

  Probiotics are usually provided by culturing specific microorganisms or, at best, a mixture of specific microorganisms. These probiotics can “normalize” the microflora when they are administered to combat enterotoxiosis conditions. In recent years, it has been found that it is possible and perhaps more preferred to provide one (or more) of the patient's own species of microorganisms. Such a scheme would fit into today's development of “individualized therapeutics”. An example is described in US Pat. No. 6,057,056, where microorganisms are isolated from human samples, cultured, and provided to the same human after sufficient culturing. In that same document, it is also proposed that recovery of the microflora, in particular the recovery of the urogenital microbiota, can be achieved with bacterial species isolated from patients. For this reason, urogenital organisms are collected and cultured, and the main healthy species are isolated and stored when the patient in need of repair or maintenance of the flora is healthy. If a human has a genitourinary microorganism that has been depleted at some later point in life, such as during pregnancy or during urogenital infection, the originally isolated organism is cultured and vaginal reimplanted or administered orally . Such an approach personalizes treatment and utilizes organisms, which are known to be associated with human health and are recognized as “self” organisms by the immune system at one stage in life.

US Patent Application Publication No. 2003 / 118,571

Teitebaum et al. 2002. Nutritional impact of pre- and probiotics as protective gastrointestinal organics. Annual Reviews Nutrition 22: 107-138 Isolauri et al. 2002. Probiotics: a role in the treatment of intensional inflection and inflation? (Gut 50: 54-59) Brandino, G.M. et al. , 2008, Probiotics: over of of microbiological and immunological charactaristics, Expert Rev. Anti Infect. Ther. 6: 497-508

  However, one disadvantage of this method is that microorganisms need to be sampled, isolated, and stored while the human is still (healthy).

  Thus, there is still a need for other methods for providing individual probiotic treatments.

The present invention
(A) obtaining a sample derived from a subject, which is obtained from a site in an enterotoxemia state and contains a microorganism;
(B) culturing the sample in vitro under conditions favorable to beneficial microorganisms;
(C) reintroducing the cultured sample of the microorganism into the subject;
Relates to a method of treating an enterotoxiosis condition in a subject comprising:

  Alternatively, the invention includes a probiotic composition for use in the treatment of an enterotoxiosis condition in a subject, wherein the composition is subject to an enterotoxiosis condition from the subject under conditions beneficial to the beneficial microorganism. It was obtained by culturing a sample containing microorganisms derived from these sites. Preferably, such a composition further comprises an excipient. More preferably, such compositions are suitable for administration to the site of enterotoxiosis conditions.

  In a preferred embodiment of the invention, the subject is a human. In another preferred embodiment, the subject is selected from the group consisting of animals, preferably cattle, horses, poultry, pigs, dogs and cats.

  In a further preferred embodiment, the site of enterotoxiosis is selected from the intestine, vagina, oral cavity, respiratory tract and skin.

  In a further preferred embodiment of the present invention, the microorganism in the sample collected from the subject and cultured is a bacterium. More preferably, the beneficial microorganism is selected from the group consisting of Lactobacillus, Ackermancia and Bacteroides.

  Also, in a preferred embodiment, the culture in vitro is maintained for more than 2 days.

  In a further preferred embodiment, the cultured cells are administered orally or topically.

  In another preferred embodiment, the cultured and concentrated sample obtained in step b) is preserved, and the sample of the preserved sample is said in the case of a recurrent enterotoxemia condition such as recurrent infection. Used to administer to a subject.

Another embodiment of the present invention is a method of suppressing the occurrence of autoimmune disease in a child born by cesarean section,
(A) collecting a maternal-derived vaginal microbiota sample at the time of delivery or immediately before or after delivery;
(B) optionally culturing the sample such that the sample can be optionally enriched for beneficial microorganisms during culture;
(C) administering at least a portion of the sample obtained from step (a) or step (b) to a newborn, preferably by oral administration;
including.

Identification of bacterial vaginitis negative (BV-) (Newgent score 0-3) and BV + (Newgent score 8-10) associated with species by culture of 16S rRNA-independent 454 pyrosequencing. Rows represent abundant genera and species in subjects, and 20 BV- and 20 BV + subjects were chosen for this study. A typical genus associated with bacterial vaginosis has been replaced by a microflora known to exist predominantly in healthy women. Data are from 2 individuals, the left side gives an overview of the bacterial species in the sample at t = 0, and the right side shows the bacterial species found after 24 hours in modified CDM medium.

(Definition)
The term “probiotic composition” as used herein refers to a composition comprising one or more acceptable excipients suitable for application to one or more probiotic organisms and mammals. . It will be appreciated that acceptable excipients will be familiar to those of skill in the art of preparing probiotic compositions. Such acceptable excipients for oral administration of probiotic compositions include, for example, sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose, sorbitol, xylitol Sugar alcohols such as erythritol, lactitol, paratinol, reduced starch syrup, reduced maltose starch syrup, polysaccharides such as maltodextrin, corn starch, rice starch, potato starch, wheat starch, fatty acid sucrose Emulsifiers such as esters, glycerin esters and fatty acids of fatty acids, thickeners (stabilizers) such as carrageenan, xanthan gum, guar gum, pectin and locust bean gum, acidifying agents such as citric acid, lactic acid and malic acid, Down juice, orange juice, fruit juices such as juices, vitamin A, vitamin B, vitamins such as vitamin C, vitamin D, and vitamin E, calcium, iron, manganese, minerals such as zinc. Compositions for topical administration of probiotic compositions additionally or alternatively include natural polymers, cellulose, including proteins such as zein, modified zein, casein, gelatin, gluten, serum albumin, and collagen, Dextran and polysaccharides such as polyhyaluronic acid, or polyphosphazenes, poly (vinyl alcohol), polyamides, polycarbonates, polyacrylates, polyalkylenes, polyacrylamides, polyalkylene glycols, polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyls Polymers such as esters, polyvinyl halides, polyvinyl pyrrolidone, polyglycolides, polysiloxanes, polyurethanes and synthetic polymers including copolymers thereof are included. Examples of suitable polyacrylates include poly (methyl methacrylate), poly (ethyl methacrylate), poly (butyl methacrylate), poly (isobutyl methacrylate), poly (hexyl methacrylate), poly (isodecyl methacrylate), poly (lauryl methacrylate) ), Poly (phenyl methacrylate), poly (methyl acrylate), poly (isopropyl acrylate), poly (isobutyl acrylate) and poly (octadecyl acrylate). Synthetically modified natural polymers include cellulose derivatives such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocellulose. Examples of suitable cellulose derivatives include methylcellulose, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethylcellulose, cellulose triacetate and cellulose sulfate. Of sodium salt. Polysaccharides such as alginate, dextran, cellulose, collagen, and chemical derivatives thereof (substitution, addition, hydroxylation, oxidation, and other modifications routinely made by those skilled in the art) such as alkyl, alkylene, and other chemical groups Degradable polymers such as, and proteins such as albumin, zein, and copolymers and mixtures thereof, alone or in combination with synthetic polymers, can also be used. Probiotics can also be administered in the form of a hydrogel. Suitable hydrogels include polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylate, poly (ethylene terephthalate), poly (vinyl acetate), as well as natural polymers such as cellulose and alginate alginate as described above. Can be formed from synthetic polymers such as copolymers and mixtures of

  Probiotic compositions for oral use may be advantageously provided in the form of dairy products such as yogurt and curd.

  Probiotic compositions for topical administration can be applied in the form of creams or ointments. Thus, they ideally contain oily substances from plant, marine or animal sources. Suitable liquid oils include saturated, unsaturated or polyunsaturated oils. As an example, unsaturated oil in any proportion is olive oil, corn oil, soybean oil, canola oil, cottonseed oil, coconut oil, sesame oil, sunflower oil, borage seed oil, clove oil, hemp seed oil, herring oil, cod liver oil , Salmon oil, linseed oil, wheat germ oil, evening primrose oil or mixtures thereof. These creams or ointments can further comprise polyunsaturated fatty acids. In one or more embodiments, the unsaturated fatty acid is selected from the group of omega-3 and omega-6 fatty acids. Examples of such polyunsaturated fatty acids are linoleic acid and linolenic acid, γ-linoleic acid (GLA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Such unsaturated fatty acids are known for skin conditioning effects that contribute to the therapeutic benefits of the composition. Thus, the composition may comprise at least 6% oil selected from omega-3 oil, omega-6 oil, and mixtures thereof. Essential oils can also be used, which are also considered therapeutically active oils, contain biologically occurring active molecules, exert therapeutic effects in topical applications, and benefit from the probiotic mixture in the composition The effect is exerted synergistically depending on the case. Another class of therapeutically active oils includes liquid hydrophobic plant-derived oils that are known to have therapeutic effects when applied topically. Silicone oils are also used and are desired due to their known skin protection and occlusive properties. Suitable silicone oils include polyalkyl siloxanes, polyaryl siloxanes, polyalkylaryl siloxanes, polyether siloxane copolymers, polydimethylsiloxane (dimethicone), poly (dimethylsiloxane)-(diphenyl-siloxane) copolymers, and the like. Contains non-volatile silicone. These are selected from cyclic or linear polydimethylsiloxanes containing from about 3 to about 9, preferably from about 4 to about 5 silicon atoms. Volatile silicones such as cyclomethicone can also be used. Silicone oils are also considered therapeutically active oils due to their barrier retention and protective properties.

  The term “enterotoxiosis” refers to microorganisms that (a) qualitative and quantitative changes in the content or amount of the microorganism itself, (b) changes in their metabolic activity, and / or (c) their local distribution. A change is defined as a condition that creates a harmful effect. In particular, vaginal enterotoxemia is defined as a health condition deviation. A healthy condition is defined in this case as a condition that has a relatively low susceptibility to sexually transmitted infections. It has been widely accepted that Lactobacillus activity contributes to maintaining this state by protecting the vaginal environment against pathogens by producing lactic acid and resulting low pH. Thus, vaginal enterotoxiosis is characterized, inter alia, by the presence of relatively high pH values.

  As used herein, the term “microorganism” refers to a symbiotic microorganism that colonizes (partially) a host organ such as the vagina, oral cavity, skin, or lungs or gastrointestinal tract with food intake, or a community of transient microorganisms. Say. However, these transient (visiting) bacteria are not considered part of the so-called “resident microbiota”.

  As used herein, “epithelium” or “epithelial cell or tissue” is one of four basic types of animal tissue that exists with connective tissue, muscle tissue, and nerve tissue. Epithelial tissue lines cavities and surfaces of structures throughout the body and forms many glands. Epithelial cell functions include secretion, selective absorption, protection, transcellular transport and sensory detection.

  As used herein, “mucosa” is a term used to indicate mucosa. Mucus (adjective form: “mucous”) is a slippery secretion produced by and covering the mucosa. Mucus fluid is typically produced from cells found in the mucus gland. Mucus cells secrete glycoproteins and water-rich products. Mucus may also be derived from a mixed gland that contains both serous and mucus cells. This includes preservative enzymes (such as lysozyme), immunoglobulins, inorganic salts, proteins such as lactoferrin, as known as mucins produced by goblet cells in the mucosa and submucosal glands, and glycoproteins It is a viscous colloid. This mucus serves to protect epithelial cells (lining the ducts) in the respiratory, digestive, urogenital, visual and auditory systems in mammals.

  In healthy animals, microorganisms are usually absent from internal tissues such as blood, brain, and muscle. However, the surface tissue, i.e. skin and mucous membranes, is in constant contact with surrounding organisms and various microbial species become easily established. The mixture of organisms normally found at any anatomical site is called the normal bacterial flora, or preferably the “resident microbiota”. The normal human flora consists of several eukaryotic fungi and protists, but bacteria are the most obvious microbial component of the normal flora. Although humans are exposed to a wide variety of microorganisms throughout their lives, only a limited number of species can permanently settle in the various body parts available, and each body part has certain characteristics. Has a microbial community of species. Living organisms at a body part grow and reproduce under the conditions that occur at that part, while those that cannot do so are considered transient. In most cases, the resident organism attaches to a substrate within the body part—this is (1) host epithelial cells, (2) extracellular matrix components (mucus), (3) another microorganism, or (4 ) A structure (eg, a tooth) produced by the host.

(Detailed Description of Embodiments of the Invention)
In cases where the patient has symptoms of enterotoxemia, treatment should focus on restoring local bacterial flora balance. Known therapies are centered on administration of cultured microbial species, where a single microorganism is prepared to restore balance. In some cases, a mixture of probiotics is available, but in most cases a single culture has been applied.

  In recent years, treatment for enterotoxiosis of the intestinal flora has become increasingly common, especially where complete samples of microorganisms are taken from healthy individuals and transplanted into the intestines of patients in need thereof. It has become. However, it is believed that there is a tremendous change from subject to subject in the composition of the bacterial flora. Microbial personalization has been demonstrated in several recent studies (eg, Faith, JJ et al., Science 341: 1123439). In these studies, strains defined as being isolated by sharing> 96% of the genomic content were considered to have been maintained over time within individuals and families, not among unrelated individuals. It was. Thus, early bowel colonization, such as that obtained from our parents and siblings, has the potential to exert physiological, metabolic and immunological effects on most, possibly all, of our lives. .

  The immune system and its relationship with mucosal tissue appears to be important in the regulation of this system. This has been demonstrated by Sharma et al. (J. Biomed. Biotechnol., 2010, 305879) who studied the function of the intestinal barrier against colonization by microorganisms. They concluded that the complex integration of this function and journal performance was performed wonderfully by the intestinal mucosa (IM) surface. Exposed to trillions of luminal microorganisms, IM is symbiotic through pattern recognition receptors (PRRs) by avoiding the threat of signaling to the innate immune system and developing tolerance (low responsiveness) to commensal bacteria Responds to bacteria. The system also works by protecting against pathogens by producing and releasing protective peptides, cytokines, chemokines and phagocytic cells. The IM always samples the lumen contents and performs molecular adjustments at its borders.

Such changes should not only be found between subjects, but can also experience significant microbial fluctuations over time in the same subject. This can be demonstrated, for example, in the vaginal microflora. The vagina, which is sterile at birth, settles the mother lactic acid bacteria within a few days. This agrees with the theory that the normal human flora is mainly derived from the mother. In most cases, this microorganism is frequent throughout life. In the vagina, its presence is mainly influenced by hormone levels. The presence of lactic acid bacteria is strongly influenced by the availability of glycogen, the level of which is regulated by estrogen. During the first few weeks of birth, there is a decrease in the population of lactic acid bacteria, which is advantageous for coagulase negative staphylococci, streptococci, E. coli and other enteric bacteria. Later in puberty, the ideal conditions for the growth of lactic acid bacteria are restored (increased estrogen levels and thickening of the vaginal mucosa). Lactic acid bacteria thus become the dominant vaginal flora of adult women. During this period, the microbial population in the vaginal secretion is about 10 ⁷ -10 ⁸ CFU / g. This microbial population remains substantially unchanged until menopause and, when replaced by more diverse microorganisms, does not differ from that of the prepubertal period, but mycoplasma and to a lesser extent anaerobic bacteria ( For example, due to the presence of a significant amount of Gardnerella vaginalis).

  Various studies have attempted to characterize vaginal lactic acid bacteria at the species level. These studies indicate that there are geographical differences in the composition of the vaginal microflora. However, in many cases, bacteria belonging to what is called the Acidophilus complex appear to constitute a dominant species at the fertility age.

  Bacterial vaginosis is an abnormal condition of the vaginal microbiota, which is characterized by increased vaginal pH, decreased Lactobacillus and increased diversity of vaginal anaerobic bacteria, Can be characterized as an example. Bacterial vaginitis is associated with adverse pregnancy outcomes, upper genital tract infections and increased risk of sexually transmitted diseases.

  Currently, methods for treating enterotoxiosis in a subject take a sample of microorganisms from the site where enterotoxiosis is present, are beneficial to the growth of beneficial bacterial species, and / or are not beneficial or A sample of said culture when these microorganisms are cultured under conditions that are detrimental to the growth of pathogenic species, and the culture is sufficiently cultured to restore a healthier balance in the presence of microorganisms Has been found to be advantageously implemented by reintroducing the same into the same subject. Of course, which micro-organisms are considered beneficial for health and which micro-organisms are not considered healthy depends on the part of the body from which the sample was taken. Taking the vagina as an example, the most abundant bacterial genus is that of Lactobacillus (Lactobacillus crispus, Lactobacillus gasseri, Lactobacillus sonar, Lactobacillus jenseny and / or Lactobacillus bajinalis), which is lactic acid Involved in production and is characterized for the vaginal environment. There are also other lactic acid-producing bacteria that contribute to the production of vaginal acids by fermentation, namely species from the genera Atopovium, Leptotricia, Leuconostoc, Megasfera, Pediococcus, Streptococcus and Weissela. It may be. However, several anaerobic bacterial species are also often found in the vagina, for example, Atopovium bajaina, Peptostreptococcus, Staphylococcus, Streptococcus, and Bacteroides, Fusobacterium , Gram-positive cocci such as Gardnerella vagina, Mobilungus, Prevotella, and Gram-negative enteric organisms such as E. coli.

  The resident microbiota of the vagina is very different from the resident microbiota that can be encountered in the skin. It is rich in Staphylococcus epidermidis, Corynebacterium, Staphylococcus aureus, Staphylococcus swaneri, Acne, Pseudomonas aeruginosa, Microgenus, Peptostreptococcus and Mycobacteria, but its presence on the skin Depending on the exact location of the hand (for example, the microflora on the back of the hand may differ significantly from the flora found on the soles).

  One site that has been mentioned in recent years to benefit from the administration of probiotics is the lung, or more generally the respiratory tract. It has recently been established that the endogenous microbiota is not only present in the upper respiratory tract, but is also established in the endogenous alveoli and bronchioles (Charson, ES et al., Am. J. Respir. Crit. Care Med. 184: 957-963, 2011). As such, they form a stable environment, which can be disturbed by pathogenic and infectious bacteria.

  A community of commensal bacteria that have settled in a part of the host organ will settle in the mucus layer and epithelial cells, which form a so-called shed surface (eg, the mammalian intestine is renewed every 4-5 days Covered with a single layer of epithelial cells). In other words, there is a certain turnover of cells settled by microorganisms in our body. Therefore, microorganisms adhering to the lumen are always released into the lumen and can be collected from sputum, saliva, feces and the like. A sample of the resident microbiota can also be obtained by taking an epithelial surface (eg, a vaginal swab).

  With regard to current treatments for bacterial vaginosis, this is currently most often achieved by administration of antibiotics such as metronidazole or clindamycin. However, these antibiotics are often ineffective, are harmful to beneficial bacteria such as lactobacilli, and are associated with a wide range of side effects and recurrence of infection. Thus, treatment of enterotoxemia caused by bacterial vaginosis with the method according to the invention is a major improvement in the treatment of bacterial vaginosis.

  In a related embodiment, culture and introduction of maternal vaginal microflora for neonatal inoculation with cesarean section appears to be advantageous. Newborns born through cesarean section are known to be prone to autoimmune diseases such as type 1 diabetes, Crohn's disease, multiple sclerosis, asthma, allergic rhinitis, atopic dermatitis and psoriasis at a later age. (Neu, J. and Rushing, J., 2011, Clin. Perinatol. 38 (2): 321-331). One of the reasons for the development of these autoimmune diseases is the “hygiene hypothesis” which suggests that an overly clean environment can contribute to the progression of the disease in some childhood periods, especially in early childhood. Explained. The latest literature suggests that the normal fetal gastrointestinal tract is sterile. During and immediately after childbirth, bacteria from the mother and the surrounding environment settle in the infant's intestines. Infants born by caesarean section have been found to have particularly few bacteria and poor diversity, even when breast-fed. Bacteroides species were deficient in such infants (Azad, MB et al., 2013, Canad. Med. Ass. J. DOI: 10.15503 / cmaj.121189). Embodiments of the present invention collect the mother's vaginal microbiota at birth, optionally culture this sample, optionally concentrate the sample in this culture, to the infant (optionally cultured, optionally concentrated) The sample (or part thereof) is orally administered.

  If the sample is taken from a site in or on the body with enterotoxemia, the sample still contains beneficial bacterial species, but they are minority and by species considered not beneficial to health Overwhelmed (eg, the two measured values on the left in FIG. 2). According to the invention, such a sample is then cultured under conditions that favor the growth of beneficial microorganisms. It will be appreciated that the determination of conditions that support the growth of beneficial microorganisms will depend on the type of bacteria that are considered beneficial for a particular part of the body. As can be seen from the above, for vaginal samples, the presence of lactic acid producing bacteria, in particular Lactobacillus, is considered healthy and therefore they will be candidates for beneficial bacteria. This means that culture conditions that favor lactic acid producing bacteria for the culture of vaginal samples will be suitable for use in the present invention.

  It is contemplated that one skilled in the art would be able to select a culture medium that would be suitable for culturing beneficial bacteria in samples taken from any part of the human or animal body. One such medium would be a medium that favors the growth of Lactobacillus species that are generally considered to be “healthy bacteria”.

  Lactobacillus MRS agar (introduced by De Man, J. et al., J. Appl. Bact. 23: 130-135, 1960) (LMRS AGAR) is a lactobacillus found in clinical specimens and dairy products and foods. Concentrated selective medium intended for isolation and culture of LMRS AGAR is a concentrated selective medium useful for the cultivation of Lactobacillus from clinical specimens and dairy and food products. The main components of this medium consist of peptone yeast extract and glucose. This medium is supplemented with sorbitan monooleate complex (a source of fatty acids) and magnesium required for additional growth. Sodium acetate and ammonium citrate are added to inhibit normal flora such as gram negative bacteria, oral flora and fungi. With both of these inhibitors added, the media has been shown to selectively improve the growth of Lactobacillus. The pH is adjusted to 6.3-6.7 to favor Lactobacillus growth. This medium is prepared, distributed, stored and packaged under anoxic conditions to prevent the formation of oxidation products prior to use.

Also, because of the growth of Lactobacillus in the vaginal flora, we used Geshnizgani A. et al. M.M. , And Anderdond A. B. , J Clin Microbiol. 1992 May; 30 (5): 1323-6, so-called CDM media was used. In this study, a chemically defined medium that simulates female genital secretions was developed for the growth of vaginal microbiota. A qualitative and quantitative study of the growth of the major components of the vaginal microflora showed that all tested vaginal isolates were able to grow in this defined medium. In order to reduce the turbidity of the medium, the CDM medium is equimolar with FeSO ₄ . Adapted by replacing hemin (source of iron ions) with 7H ₂ O.

  As shown in the experimental section, we established growth against lactobacilli as a result of acidification of the medium from lactate production during incubation with the vaginal flora for 24 hours ( Lactobacilli are very acid resistant).

  Next to the Lactobacillus species, depending on the part of the body from which the sample was taken, other bacteria could be considered beneficial, and selective culture media were developed for the cultivation of such bacteria. obtain. An example of such other microorganisms is Ackermancia (eg Ackermansia muciniphila), which has recently been established to be predominant and abundant in the gastrointestinal tract and is beneficial for the prevention of obesity (Derrien M. et al., Int. J. Syst.Evolut.Microbiol.54: 1469-1476, 2004; Everard, A. et al., Proc.Natl.Acad.Sci. ). A specially developed medium for growing Ackermansia mushini Phila has been described by Derrien et al. , 2004.

  Depending on the origin of the microbial sample, the culture may contain some or many transient microorganisms. These transient microorganisms are not part of the “resident microbiota” and should preferably be excluded. In some cases, it is complex to distinguish between transient and resident microorganisms for parts of the body that contain a large number of transient microorganisms (eg, the digestive tract). One criterion that can help distinguish (and separate) these transient and resident microorganisms is that they cannot adhere to human tissue.

Thus, elimination of transient bacteria can thus be achieved by the following methods alone or in combination. These methods can be used in addition to or in place of the methods described above for culturing microbial samples.
(I) Use of a medium very similar to the conditions of the body part from which the sample originated. Conditions similar to the normal environment for the resident microbiota are presented to favor their growth compared to microorganisms that exist only transiently. Some evidence has recently been established in a dynamic intestinal model that specific culture conditions similar to local body conditions benefit the resident microbiota, where they incorporate a mucosal environment Has been demonstrated to result in enrichment of samples for lactobacilli (Van den Abbeele P, et al., Microb Biotechnol. 2012 Jan; 5 (1): 106-15. Doi: 10.11111 / j.1751 -7915.20111.00308.x.Epub 2011 Oct 12).
(Ii) separating resident microorganisms from transient microorganisms by assaying their ability to bind to mucosa and / or epithelium. Such a method is described in EP 1995535 dealing with the culture of Lactobacillus acidophilus. Assays for detecting these types of bacteria are described in Fakry, S .; et al. , Res Microbiol. 2009 Dec; 160 (10): 817-23. doi: 10.1016 / j. resmic. 2009.9.09.009. Epub 2009 Sep 24).
(Iii) As described above in item (ii), as well as screening for mucosal or epithelial binding ability, such as mucus-binding protein, or made possible by the use of available antibodies Screening for presence by hybridization to genes or PCR-based experiments can be used. Many genes and proteins are known and they are part of many families of genes and proteins characterized by having a consensus sequence, particularly a functional part of the protein, ie it binds to mucosal tissue or epithelium It is a part to do. For example, one of the most abundant consensus sequences is the so-called LPXTG domain that is characteristic of cell anchors.

  After sufficient culture, the culture (a portion) may be prepared for reintroduction into the patient, but the culture or the rest of it may be preserved. Preferred storage conditions are storage at a very low temperature (−80 ° C.). In such cases, a cryoprotectant should be added to the medium (eg, 15% glycerol) as is common in the art. Storage of the culture allows maintenance of the bacterial stock culture from which the sample can be thawed many times and used as an inoculum for new cultures. To. This allows for long-term supply of cultures and multiple reintroductions

  If the sample is well cultivated, i.e. there is a sufficient amount of beneficial bacteria available in culture for non-beneficial bacteria, the sample is reintroduced into the body part from which it was originally derived. The This reintroduction can be done by any available method or using any available formulation of the cultured sample.

  The cultured sample may be reintroduced by formulating it as a probiotic composition. A probiotic composition as defined herein can take any form. Where appropriate (eg in the case of intestinal flora) it can be administered orally, preferably topically applied. When applied topically, it may be in pure form, but it can also be applied in the form of a cream, foam, lotion or ointment. Topical application may include application by inhalation, which may be advantageous when, for example, the culture sample is reintroduced into the oral cavity or applied to the respiratory tract including the lungs. For reintroduction into the oral cavity, it can be applied via other oral dosage forms that disintegrate in the oral cavity, such as the activity of amylase in saliva.

  As will be appreciated by those skilled in the art, the reintroduced probiotic composition may contain excipients that may be necessary or advantageous for the administration of bacterial cultures. Many excipients have already been described in the definition of the probiotic composition of the present invention, but this list includes any excipient, carrier or carrier used in a pharmaceutical or dietary supplement composition. Can be extended with additives. Very useful dairy products, often used as media for classical probiotics, can be used for delivery of the probiotic compositions of the present invention to the oral cavity or gastrointestinal tract.

  The second embodiment of the present invention involves the treatment of recurrence of bowel disease, specifically Clostridium difficile infection. In this case, treatment with antibiotics including vancomycin and metronidazole is also associated with frequent recurrence. This probably occurs as a result of a persistent decrease in Clostridium difficile spores that cause colonic microbiota protective effects and reinfection. As another strategy, fecal flora transplantation (FMT) has been successfully applied to restore healthy intestinal flora in the patient's infected colon. As already indicated in the background section, this treatment strategy benefits from the ability to settle in a host of beneficial bacteria when the microbiota donor is a close relative of the patient. Cultivation and storage of fecal flora from patients taken prior to recurrence of infection provides advantages over current methods to overcome the complex logistic associated with cost and fecal transplantation. Also, sampling is done only at the time of infection, which means it is done when treatment is needed. The fact that cultured (and concentrated) samples can be stored and used as new inoculum at the time of recurrence of infection is an advantage that should not be ignored. In many cases, the initial anti-infective treatment is only partially effective and the method of the invention will be a welcome additional therapy to help overcome the infection. However, even with such additional therapies, there is a risk of recurrence, after which a stored sample of concentrated microorganisms will be useful.

  It should be noted that the method of the present invention is truly individualized, but the method used is general for each medical application. The principle is based on the selective enrichment of individual beneficial bacteria from a collection of bacteria from a specific body part. Subsequent administration of the concentrated culture is postulated to be much more effective in restoring a healthy microflora than the probiotic supplements available today.

  However, it is possible to have improved generalization. In such cases, (enriched) samples of multiple subjects are collected and further co-cultured to grow relatively typical microorganisms for all subjects. Such “generalized” expanded microbial populations are less individualized than the cultures obtained by the methods described above, but are sufficiently effective for the majority of people. In particular, when enterotoxemia is acute and progressing rapidly, that is, when there is not enough time to obtain a sample and culture as described above, such “general” The possibility of having a "made up" sample is very valuable and can at least minimize the symptoms caused by enterotoxemia in particular.

  Although the above has been described with particular emphasis on the human situation, it is also proposed that the present invention can be used advantageously for veterinary applications. Of particular interest are intestinal health, oral health and vaginal health applications in all economically important species such as cattle, horses, poultry, pigs, dogs, cats and the like. Two specific examples of such applications are intestinal health applications in broilers (including spawning hens and embryos in eggs) and intestinal health applications in pigs and piglets. In both cases, lactic acid bacteria dominate the gut microbiota and are thought to be associated with a positive direction for health. Also, only a few lactic acid bacteria species are involved, which means that these examples are very similar to the human vaginal situation as described above (and in the examples) To do.

  The invention is further illustrated by the following examples. The examples should in no way be construed as limiting the scope of the invention.

(Example 1: Concentration of vaginal microflora)
(sampling)
In women with high-risk sexual behavior, routine screening for sexually transmitted diseases (STD) in the clinic involves standard cervical examinations. During a normal sampling, the mucus was collected using a cotton swab and resuspended in a tube labeled screw cap with AMIES transport medium (plus 15% glycerol and cysteine solution added to it). . Immediately thereafter, the tube was placed in liquid nitrogen and transferred for storage at -80 ° C. Gram staining, Amsel criteria, and STD status are obtained at the clinic as usual for normal purposes. The prevalence of bacterial vaginosis (BV) was usually observed in 50% of a sampled population of women with high-risk sexual behavior.

  Nugent scoring was performed on all samples prior to storage. Nugent scoring is a Gram stain scoring system and is used for the Papanicolaou test to diagnose BV. First described by P Nugent in 1991 (J. Clin. Microbiol. 29: 297-301, 1991). Nugent score: large gram positive gonococci (Lactobacillus form type; Lactobacillus reduction scored as 0 to 4), small Gram indeterminate gonococcus (Gardnerella vaginal form type; scored as 0-4), and curved gram It is calculated by evaluating against the presence of indeterminate Neisseria gonorrhoeae (Mobilungus genus type; scored as 0-2) and can range from 0-10. A score of 7-10 is consistent with BV.

  In this study, samples with a neutral score in the range of 8-10 were selected. And as a control, samples of “health” with a nugent score of 0-3 were included.

(concentrated)
The screw cap tube containing the resuspended vaginal swab was removed from the −80 ° C. freezer and thawed at room temperature under anaerobic conditions. The suspension volume 130 [mu] L, of 13mL in (modified by the substitution of hemin by FeSO ₄ .7H ₂ O in an equimolar amount in order to reduce the turbidity of the medium (a source of iron ions)) Corning tube 50mL It was transferred to denatured CDM medium (Geshnizani, A. et al., J. Clin. Microbiol. 30 (5): 1323-1326, 1992). Thereafter, the cells were cultured for 24 hours at 100 rpm and 37 ° C. under anaerobic conditions.

(Determination of (enriched) microbial species by barcode 16S-amplicon sequencing)
Prior to the construction of a 16S rRNA gene amplicon library spanning the variable region V5-V7 (Bogaert, D. et al., PLoS One 6 :: e17035, 2011), quantitative 16S-PCR was performed and isolated DNA The relative amount of bacterial template in the sample was determined (Biesbroek, G. et al. PLoS One, 7: e32942, 2012). Sequence analysis of the amplicon library was performed with a 454 GS-FLX-titanium sequencer (Life Sciences (Roche), Branford, CT). FASTA-formatted sequences and corresponding quality scores were generated with a GS-FLX titanium sequencer using the GS amplicon software package (Roche, Branford, CT). Extracted from the sff data file, 1.22.2 Software platform implemented using modules (Schloss, P. et al., Appl Environ Microbiol 75: 7537-41, 2009). Sequences were denoised using a pseudo-single chain algorithm (Huse, S. et al., For the purpose of removing sequences that tend to be due to pyrosequencing errors ("pre.cluster" command). Environ.Microbiol.12: 1889-1898, 2010). Potential chimeric sequences were detected and removed using the “chimera.uchime” command (Edgar, R. et al., Bioinformatics 27: 2194-2200, 2011). High quality aligned sequences were classified using the RDP-II naive Bayes classification (Wang, O. et al., Appl Environ Microbiol 73: 5261-5267, 2007). The aligned sequences were clustered into OTUs (defined by 100% similarity) using an average linkage clustering technique.

(result)
The presence of genus related to BV (including Gardnerella, Bacteroides, Parvimonas, Atopobium, Sneatia, Prevotella, Dialista, and Megasfera) in samples with a neugent score of 8-10 by sequence analysis of 454, and new of 0-3 The presence of species related to “health status” in the past with a gent score (including Lactobacillus spruce, Lactobacillus gasseri and Lactobacillus enseny) has been confirmed. This confirmed the survey results described in the past (Ravel et al, PNAS Early Edition, 108: 4680-4687 2010).

  The method of concentrating BV microbiota resulted in suppression of BV related microbiota and accumulation of lactobacilli. For example, in some cases, an increase in total lactic acid bacteria of 18% to 96% was obtained (see below for a summary of the most abundant species table; all tables show two replicates at t = 0 and t = 24 including).

(Appendix)
(Appendix 1)
(A) obtaining a sample derived from a subject, which is obtained from a site in an enterotoxemia state and contains a microorganism;
(B) culturing the sample in vitro under conditions favorable to beneficial microorganisms;
(C) reintroducing the cultured sample of the microorganism into the subject;
A method of treating an enterotoxiosis condition in a subject comprising:
(Appendix 2)
Obtained by culturing a sample containing microorganisms from a site of enterotoxiosis conditions from a subject under conditions beneficial to beneficial microorganisms;
A probiotic composition for use in the treatment of an enterotoxiosis condition in a subject.
(Appendix 3)
Further comprising an excipient,
The probiotic composition according to Supplementary Note 2, wherein
(Appendix 4)
The composition is suitable for administration to a site of enterotoxiosis conditions,
The probiotic composition according to Supplementary Note 3, wherein
(Appendix 5)
The subject is a human;
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 6)
The subject is selected from the group consisting of animals, preferably cattle, horses, poultry, pigs, dogs and cats,
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 7)
The site of enterotoxiosis is selected from the intestine, vagina, oral cavity, respiratory tract and skin;
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 8)
The microorganism in the sample is a bacterium;
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 9)
The beneficial microorganism is selected from the group consisting of Lactobacillus, Bifidobacteria and Ackermancia,
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 10)
the culture in vitro is maintained for more than 2 days,
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 11)
The reintroduction is performed by oral administration or topical administration.
The method according to supplementary note 1 or the probiotic composition according to supplementary note 2, characterized in that:
(Appendix 12)
The cultured and concentrated sample is stored, and a sample comprising the stored sample is used for administration to the subject in a case of a recurrent enterotoxiosis condition such as a recurrent infection. the method of.
(Appendix 13)
(A) collecting a maternal-derived vaginal microbiota sample at the time of delivery or immediately before or after delivery;
(B) optionally culturing the sample such that the sample can be optionally enriched for beneficial microorganisms during culture;
(C) administering at least a portion of the sample obtained from step (a) or step (b) to a newborn, preferably by oral administration;
To suppress the occurrence of autoimmune diseases in children born by caesarean section.

Claims (13)

(A) obtaining a sample derived from a subject, which is obtained from a site in an enterotoxemia state and contains a microorganism;
(B) culturing the sample in vitro under conditions favorable to beneficial microorganisms;
(C) reintroducing the cultured sample of the microorganism into the subject;
A method of treating an enterotoxiosis condition in a subject comprising: Obtained by culturing a sample containing microorganisms from a site of enterotoxiosis conditions from a subject under conditions beneficial to beneficial microorganisms;
A probiotic composition for use in the treatment of an enterotoxiosis condition in a subject. Further comprising an excipient,
The probiotic composition according to claim 2. The composition is suitable for administration to a site of enterotoxiosis conditions,
The probiotic composition according to claim 3. The subject is a human;
3. The method according to claim 1 or the probiotic composition according to claim 2. The subject is selected from the group consisting of animals, preferably cattle, horses, poultry, pigs, dogs and cats,
3. The method according to claim 1 or the probiotic composition according to claim 2. The site of enterotoxiosis is selected from the intestine, vagina, oral cavity, respiratory tract and skin;
3. The method according to claim 1 or the probiotic composition according to claim 2. The microorganism in the sample is a bacterium;
3. The method according to claim 1 or the probiotic composition according to claim 2. The beneficial microorganism is selected from the group consisting of Lactobacillus, Bifidobacteria and Ackermancia,
3. The method according to claim 1 or the probiotic composition according to claim 2. the culture in vitro is maintained for more than 2 days,
3. The method according to claim 1 or the probiotic composition according to claim 2. The reintroduction is performed by oral administration or topical administration.
3. The method according to claim 1 or the probiotic composition according to claim 2.   2. The cultured and concentrated sample is stored, and a sample comprising the stored sample is used for administration to the subject in a case of recurrent enterotoxiosis such as a recurrent infection. The method described. (A) collecting a maternal-derived vaginal microbiota sample at the time of delivery or immediately before or after delivery;
(B) optionally culturing the sample such that the sample can be optionally enriched for beneficial microorganisms during culture;
(C) administering at least a portion of the sample obtained from step (a) or step (b) to a newborn, preferably by oral administration;
To suppress the occurrence of autoimmune diseases in children born by caesarean section.